The overall aim of our research is to develop a combined engineering, ultrastructural and biophysical approach to the mechanisms whereby endothelial cells and the clefts between the cells modulate microvessel permeability. Freeze fracture studies and ultrathin serial sections have demonstrated that endothelial cells are joined by an array of junctional strands which are interrupted at intervals allowing passage of water and solutes. Cytochemical and permeability studies also indicate that all or part of the cleft may be filled with a fibrous matrix. We describe novel experiments guided by a new conceptual theoretical framework to relate permeability properties of segments of individually perfused microvessels, having known permeability properties, to the ultrastructure of the junctional strands between adjacent endothelial cells and a fiber matrix within the cleft. The specific aims of the proposal are 1) to evaluate the proposal that the permeability properties of microvessel walls to water and small solutes are determined by the frequency of small discontinuities in the junctional strand; and 2) to evaluate the relative contribution of structures associated with the junctional strand and the fiber matrix to the molecular filter at the microvessel wall. The primary focus of our new approach is the analysis of the three-dimensional convective-diffusive spread of tracer molecules on the albuminal side of the interruptions in the junction strand arrays. This spread is analogous to the growth of a wake on the downstream side of a small object at very low Reynolds numbers. Preliminary results from serial sections by Dr. Adamson indicate that this approach is necessary to investigate the geometry and distribution of hypothetical small pores or slits in the junction strands which cannot be resolved by conventional 30-40nm sections. The combined theoretical and experimental approach adopted herein takes advantage of a recently developed highly accurate analytic solution by Dr. Weinbaum for the three-dimensional viscous flow in a cleft, which contains junction strands with discrete pores and slender cross-bridging fiber components. The model is needed to interpret the three-dimensional distribution of tracer in the wake experiments proposed in Specific Aim 1, and to analyze the results of the experiments with larger solute molecules proposed in Specific Aim 2. In both experimental designs measurements will be performed on individual perfused microvessels of precisely known permeability properties using techniques that have been extensively developed in Dr. Curry's laboratory. We believe this combined theoretical and experimental approach goes far beyond past and current attempts to delineate the junction and fiber matrix structures which modulate microvessel permeability. These studies are the most direct approach to a new understanding of the nature of the resistances which determine microvessel permeability in normal tissue and during recovery from injury.